Throughout this application various publications are referenced by arabic numerals within parantheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully described the state of the art to which this invention pertains.
The isolation of mutant clones from cultured mammalian cell lines usually relies on the application of a selective growth condition under which the mutant cells can grow while the wild type cells cannot. In some cases, however, the mutant phenotype does not present an obvious growth advantage that can be exploited toward this end. An alternative method for recognition of mutant cells has been the differential staining of mutant colonies under conditions where the cells remain viable (e.g., the immunoprecipitation of secreted products into a surrounding agar medium (1, 2)). Often the staining procedure involves cell fixation; in these cases sib selection (3) or replica plating (4, 5) has been used to isolate the viable mutant cells. In certain of these prior methods a portion of the mutant cells are exposed to conditions under which the desired mutant cells cannot survive. By killing a portion of the mutant cells the occurrence of the desired mutation is confirmed. The advent of the fluorescence-activated cell sorter (FACS) has provided a less costly, more convenient and more direct way to recover mutant cells from amongst much larger populations of mutagenized cells than can be screened at the colonial level. Provided that the mutant cells can be distinguished from wild type with a fluorescent reagent, large numbers (millions) of individual cells, rather than colonies, can be screened. The sorted population that results can be greatly enriched in mutant cells. FACS sorting has been applied primarily to the isolation of cell populations of different development status. Most mutants or variants that have been isolated with this technique have been those affected in the expression of cell surface proteins (see ref. 6 for a review). One exception has been the selection of non-clonal subpopulations of a mouse hepatome cell line exhibiting increasd or decreased rates of aromatic hydrocarbon metabolism (7). In one embodiment of the invention described herein, the FACS technique has been applied for the isolation of mutant mammalian cells that are devoid of dihydrofolate reductase, an internal enzyme.
The isolation by a selective matabolic method of mutant Chinese hamster ovary (CHO) cells lacking dihydrofolate reductase (DHFR) activity, i.e. lacking a functional dhfr gene, has been described previously (8, 9). There, the mutant cells were obtained form CHO cells which were functionally heterozygous with respect to the dhfr gene. Under the appropriate growth conditions DHFR-deficient cells are perfectly viable. Moreover, they can be selected from amongst large populations of functionally heterozygous wild type cells by killing the latter with a tritiated deoxyuridine suicide technique (8). While it is expensive and labor intensive, this selective growth technique works well for selecting mutants from mutagenized populations where their frequency is higher than 10.sup.-6. However, because even moderate cell densities interfere with this selection (10), a large number of cells must be employed at a low cell density. Thus the selective growth technique represents a relatively costly and laborious procedure for the selection of spontaneous mutants, which occur at a frequency on the order of 10.sup.-7. Accordingly, the selective growth technique would be impractical for recovery in a single step of mutant cells containing a double mutation, e.g. for recovering mutant cells lacking DHFR activity (i.e. lacking a single functional dhfr gene) directly from mutagenized wild type cells which are functionally deploid with respect to the dhfr gene (i.e. containing two functional dhfr genes). Such an approach would certainly be impractical and most likely unsuccessful based on previous attempts (8). Therefore, the practical utility of the selective growth method depends on the availability of wild type cells which are functionally hemizygous or heterozygous with respect to the gene to be mutated. Often such cells are not available. Moreover, the isloation of such cells by selective growth methods is extremely difficult, requiring additional manipulations, and has not proven to be readily reproducible.
A FACS technique was therefore investigated as a more practical, more reliable and more widely applicable means of selecting or enriching for mutants which are either devoid of DHFR activity, i.e. contain no functional copy of the gene, or are functionally hemizygous or heterozygous with respect to the dhfr gene, i.e. contain one functional copy of the gene. Kaufman et al. (11) had shown that a fluroescent derivative of methotrexate could be used in the analysis and separation of cells that contained elevated levels of DHFR due to gene amplification. Methotrexate is a substrate analog that binds to DHFR with high affinity. After treatment with fluoresceinated Methotrexate, the stoichiometric binding of the drug provides an accurate indicator of the number of DHFR molecules per cell (11). Preliminary experiments by others had also indicated that the low level of fluorescence yielded by wild type CHO cells carrying two diploid copies of the dhfr gene could be distinguished from the background value exhibited by a mutant lacking DHFR activity, i.e. carrying no function copies of the dhfr gene (8). The aforementioned preliminary experiments did not involve CHO cells carrying a single copy of the dhfr gene, i.e. functional hemizygotes or heterozygotes, and no mutant cells were recovered. In the present invention, mutant cells containing no copy, a single copy or two copies of a gene, e.g. the dhfr gene, can be distinguished by the FACS. This instrument can be used by the method of this invention to recover mutant cells containing a single functional gene copy as well as mutant cells devoid of activity associated with the gene, i.e. containing no functional copy of the gene.